Salmonella is the primary exemplar of a foodborne enterobacterium studied in medical research. Nevertheless, for over one thousand of its genes, functions have not yet been characterized, and new functions continue to be found for genes that were previously characterized. Many genetic determinants of survival in countless environments are still unknown. To address this knowledge gap, we have developed several resources in the past, including complex pools of random transposon insertion mutants and collections of genome-wide systematic defined single-gene deletion (SGD), and multi-gene deletion (MGD) mutants in the most studied pathogenic strain of Salmonella, S. enterica sv Typhimurium 14028s. The collections have been used by many researchers worldwide, and numerous publications prove their utility and value. With the incorporation of of high-throughput sequencing (HTS), screening of pools has become even more popular. Pools of mutants can be used to identify gene requirements for survival of a bacterium in any environment, but only if the number of mutants in a pool is lower than the number of founder bacteria that get through biological barriers prior to reaching that environment. Otherwise, mutants may be lost by chance rather than due to a difference in fitness when screened. This is a crucial caveat, since founder populations of Salmonella are often small during infection in animals and plants. Therefore, collections of defined deletion mutants, where the maximum number of genes can be screened with a minimum number of bacterial clones, are highly sought after. In this project, we will vastly increase the value of our previous SGD and MGD collections by introducing unique 21 base DNA barcodes, flanked by Illumina sequencing primers, into each mutant. Without barcodes, characterization of pools of mutants requires multiple complex steps. With barcodes, direct PCR amplification of the barcoded region from a single-tube crude extract of bacterial cells generates a library ready for sequencing. Consequently, the use of barcodes vastly improves on older methods, by increasing throughput, saving time and costs, and by generating more accurate and robust data that effectively mitigates against partial compensatory mutations. Recognizing these crucial advantages, we have been encouraged by over 40 leaders in Salmonella research to transform our existing defined mutant collections into barcoded resources. In this proposal, we have devised a bulk strategy to build a barcoded SGD and MGD resource at low cost, by maximizing the efficiency of mutant construction and mapping through a combination of robotics, strategic pooling, and HTS. The manufacturing process also introduces an alternative antibiotic resistance, which simplifies downstream assembly of multiple mutations into a single strain by sequential transduction into a wild type strain. Just like the previous collections, the barcoded resource will be submitted to a public repository.